Novel lymphokine for suppressing platelet activation

ABSTRACT

New lymphokine and its isolation and purification process. Said lymphokine is comprised of a factor obtained from T cells stimulated by concanavaline A or by an antigen capable of inhibiting the IgE-dependent platelet citotoxicity with respect to young larvae of S. Mansoni, of strongly reducing the chemiluminescence of blood platelets in a reaction IgE-anti-IgE, which is a correlate of the anti-parasite cytotoxicity, and of inhibiting the platelet activation in non-IgE dependent intolerences. Application as suppressor agent for suppressing platelet activation and as immunomodulator medicament of allergies.

BACKGROUND OF THE INVENTION

The present invention relates to a novel lymphokine which suppressesplatelet activation, to its process of isolation and purification and tomedicaments containing it.

It is known that stimulated T cells excrete a certain number of solublefactors among which certain are capable of regulating the functions ofeffector cells.

It has been demonstrated recently (cf. M. JOSEPH, C. AURIAULT, A.CAPRON, M. VORNG and P. VIENS, NATURE 303:8IO) that blood plateletstaken up in rats infected with Schistosoma mansoni, express antiparasiteproperties destructive in vitro, which increase in the course of theinfection. The maximum cytoxicity has been observed when the rats haveexpressed a high level of immunity with respect to re-infection. Thisplatelet cytoxicity declines rapidly after eight weeks of infection,despite the presence of IgE antibodies in the serum of these infectedrats. This is the reason why the inventors have been led to considerthat the factors of the T cells, produced after stimulation ofsuppressor T cells by circulating antigens of S. mansoni, play a role inthis diminution, and this mechanism can appear as a regulation in returnof immune functions of the platelets in the infection of the rat. In thesame way, it has been shown that human platelets can be induced incytoxic effectors against schistosomules, by incubation with serum richin IgE of patients afflicted with schistosomiasis or asthmatic allergypatients, the cytoxic process being retarded in the latter case by theaddition of anti-IgE or of a specific allergen (cf. M. JOSEPH, J. C.AMEISEN, J. P. KUSNIERZ, V. PANCRE, M. CAPRON and A. CAPRON, 1984, C. R.ACAD. SC. PARIS 298:55).

It is an object of the present invention to isolate a suppressor factorof the activity of the blood platelets, which is capable of playing,among other things, an immunomodulator role of allergic manifestations.

The present invention relates to a factor obtained from T cellsstimulated by Concanavaline A or by an antigen, which can inhibit theIgE-dependant platelet cytotoxicity with respect to young larvae of S.mansoni, of greatly diminishing the chemoluminescence of platelets in anIgE-anti/IgE reaction, which is in correlation with the antiparasiticcytotoxicity and of inhibiting platelet activation in non-IgE dependantintolerances, which factor is denoted by the name of lymphokine for thesuppression of platelet activation (LSPA) and is produced by T OKT8⁺lymphocytes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the percentage inhibition of the cytotoxicity as a functionof the amount of Concanavaline A used as a stimulant.

FIG. 2 shows the percentage inhibition of the cytotoxicity as a functionof the amount of supernatant employed.

FIG. 3 shows the percentage inhibition of the cytotoxicity representedas a function of the addition of supernatant in time, expressed inhours.

FIG. 4 shows the effect of the supernatant of T lymphocytes stimulatedby Concanavaline A on the chemoluminescence of platelets.

FIG. 5 illustrates the level of respective suppressor activity of theLSPA obtained from T cells, from OKT8⁺ OKT4⁻ cells and from OKT8⁻ OKT4⁺cells, stimulated by Concanavaline A.

FIG. 6 illustrates the percentage inhibition of the cytotoxicity ofsupernatants treated by trypsin, protease K and neuraminidase.

FIG. 7 shows a chromatographic profile of the inhibitor lymphokine ofblood platelets according to the present invention.

FIG. 8 shows the percentage inhibition of cytotoxicity as a function ofpH.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In one embodiment of LSPA, the latter is isolated from supernatants TOKT8 lymphocyte cultures stimulated by Concanavaline A or an antigen.

In another embodiment of LSPA, in the case where it is isolated fromsupernatants of T OKT8⁺ lymphocyte cultures stimulated by an antigen,the T lymphocytes have been stimulated by Echinococcus granulosus orSchistosoma mansoni antigens. The present invention also relates to aprocess of preparation of LSPA which consists of stimumlating T cells ofhuman or murine origin with Concanavaline A or of a similar mitogenicstimulant or by an antigen taken from the group which comprises antigensof S. mansoni and antigens of Echinococcus granulosus, cultivating thestimulated T cells, for a suitable period, recovering the culturesupernatants and filtering them, if necessary after centrifugation,through membranes of pore dimension of the order of 0.22 μm, thenpurifying them by gel filtration and subjecting the active proteinfraction collected, previously identified by research of the biologicalor immunological activity and if necessary freeze-dried, to reversephase chromatography on bonded silica gel, of calibrated granulometryand porosity, followed by elution by a gradient of CH₃ CN--H₂ Ocontaining 1% of trifluoroacetic acid, ranging from 1-99 to 60-40, thepurified protein fraction containing the LSPA being then advantageouslyfreeze-dried after detection by recording optical density at 215 nm anddetermination of the biological activity.

In an advantageous embodiment of the process for preparing the LSPAaccording to the present invention, the biological activity of theactive protein fraction and of the purified active protein fractioncontaining the LSPA is determined by obtaining 60% inhibition of thecytoxicity by the culture supernatant of T lymphocytes stimulated byConcanavaline A or by antigens of E granulosis, with respect to normalblood platelets used as effector cells, incubated with serum of patientsafflicted with schistosomiasis or with serum of allergic patients havinga high level of circulating IgE and stimulated by anti-IgE or by thespecific allergen.

It is also an object of the present invention to provide a novelmedicament for immunomodulating allergic manifestations which ischaracterized in that its active constituent is LSPA isolated fromculture supernatants of T lymphocytes stimulated by a mitogenic agentsuch as Concanavaline A or the like or by a suitable antigen such asparticularly an E granulosis or S mansoni antigen, which has theproperties mentioned above and whose molecular weight is of the order of15-20 kDa and Pi of the order of 4.4 to 5.0, which lymphokine possessesa specific linkage structure of the type of a receptor, at the surfaceof the blood platelets, that is to say a specificity of adsorption onthe platelets.

The tests carried out to check the biological activity of the culturesupernatant of T lymphocytes stimulated according to the presentinvention have permitted arrival at the following conclusions:

Effect of the supernatant on the cytotoxicity of the platelets

The supernatants obtained after the stimulation of normal human,peripheral or amygdal T lymphocytes, by Concanavaline A (0.01 at 5 μg/ml) or T lymphocytes of the spleen of a patient afflicted withEchinococcosis, by E granulosis antigens (5 to 100 μg/ml), tested incytotoxic processes of platelets with respect to schistosomules, havepermitted results collected in the following Table 1 to be obtained:

                  TABLE I                                                         ______________________________________                                        Effect of the LSPA on the IgE dependant                                       anti parasitic cytotoxicity of murine and human                               blood platelets derived from infected and allergic                            individuals                                                                                 Man (.sup.a)                                                    Cytoxicity    A          B         Rat (.sup.b)                               ______________________________________                                        In an activator medium                                                                      72.7 ± 2.9                                                                            81.7 ± 5.5                                                                           71.2 ± 5.2                              with Con A supernatant                                                                      20.5 ± 2.1                                                                            22.1 ± 4.0                                                                           19.5 ± 2.5                              with AG supernatant                                                                          35.0 ± 15.0                                                                          33.6 ± 6.1                                                                           21.5 ± 0.5                              with lymphocyte culture                                                                     70.2 ± 3.2                                                                            81.5 ± 4.6                                                                           78.0 ± 7.0                              ______________________________________                                         N.B. In all cases the cytotoxicity with normal human or rat serums, does      not exceed 9.5 ± 7.3                                                  

a) The purified platelets were incubated with A serum (of patientsafflicted with Schistosomiasis mansoni) or B (of allergic donors) in thepresence of larvae of S mansoni (Schistosomules). The supernatant oflymphocytes or culture media of lymphocytes and anti-IgE's orcorresponding allergen in the case of incubation in serum of allergicdonors, were added and the cytotoxicity was evaluated as a percentage ofschistosomules dead after a period of incubation of 24 hours at 37° C.(mean±standard deviation).

b) The purified platelets were incubated with serum of rats afflictedwith Schistomiasis mansoni in the presence of larvae of S.mansoni(schistosomules). The supernatant of lymphocytes or the culture mediumof lymphocytes was added and the cytotoxicity was evaluated as apercentage of schistosomules dead after a period of incubation of 24hours at 37° C. (mean±standard deviation).

The Inventors have established that the supernatants are active directlyon the platelet functions and not by protection of the targets, sincethe separate preincubation of the effector platelets or of theschistosomules with the supernatants of T lymphocytes, followed by acytotoxicity test in which the platelets and the parasites have beenseparated by a membrane of polycarbonate of porosity 0.2 only ends theexpected inhibition in the case where the platelets have beenpreincubated and not in the case where the larvae have been incubated,as emerges from Table 2 below:

                  TABLE 2                                                         ______________________________________                                        Direct action of the LSPA on the human blood platelets                                    P/S     P*/S      P/S*                                            ______________________________________                                        Cytoxicity IgE/anti-IgE                                                                     92.3 ± 5.1                                                                           14.6 ± 9.2                                                                           71.5 ± 6.3                               ______________________________________                                    

The human platelets (P) and the schistosomules (S), treated (*) or notby a supernatant of lymphocytes, have been separated by a filter (/) ofpolycarbonate of porosity 0.22 μm in Boyden chambers.

The cytotoxicity was evaluated as a percentage of schistosomules dead atthe end of an incubation period of 24 hours at 37° C. (mean I.S.D.).

Moreover, the Inventors have demonstrated that this inhibition is notthe consequence of a toxic effect of a supernatant of T lymphocytessince LDH has not been detected in the platelet suspension(Lactico-dehydrogenase).

Mitogenic stimulation of T lymphocytes of spleen of rats either byConcanavaline A or by PHA ((2 μg/ml); PHA=Phytohemagglutinin) or theantigenic stimulation of mesenteric T lymphocytes or of rat spleeninfected with S. mansoni, by the antigen of adult schistosoma producedfrom supernatants which inhibit the IgE-dependent cytotoxicity ofplatelets of rats (40 to 60% inhibition), which shows that the Tlymphocytes of rats secrete, in the same way as human T lymphocytes,LAPS lymphokine, which is a platelet inhibitor factor.

As is seen in the accompanying FIG. 1, which represents the percentageinhibition of the cytotoxicity as a function of the amount ofConcanavaline A (expressed in μg/ml) used as a stimulant, the inhibitoreffect is proportional to the dose of Concanavaline A used for thestimulation of T cells and the optimal effect is obtained at a finaldose of 1 μg/ml. The supernatants produced in the absence ofConcanavaline A also express a suppressive activity, but substantiallyless than that observed with supernatants activated with lectine(20-25%).

As is seen in FIG. 2, which shows the percentage inhibition of thecytotoxicity as a function of the amount of supernatants (expressed inμl) employed, the supernatant acts in a manner which is the function ofthe amount employed to to reach a plateau at 50 μl (1:4 of finaldilution), which is the dose used for all subsequent experiments. Theeffect of the addition of supernatants at different periods before andafter the commencement of the cytotoxic process is shown in FIG. 3attached in which the percentage inhibition of the cytotoxicity isrepresented as a function of the addition of supernatant in time,expressed in hours; the optimal inhibition has been obtained by theaddition of T cell supernatant in the course of the first hours ofcontact between the effectual platelets and the schistosomules (in thisfigure "Smules" indicates schistosomules);

Structure of linkage to the platelets

Three successive incubations of supernatant of T cells which contain thefactor according to the invention, which is capable of inhibiting thedestructive properties of the platelets with respect to schistosomules,with platelets put into tablets, eliminate its inhibitor activity fromthe cytotoxicity of the platelets: this absorption of the suppressorfactor by the platelets suggests the existence of a liaison structure.On the other hand, when there are used cell lines of IgE myelome, ofK562, of U937 or of broncho-alveolar macrophages as absorbants, theinhibitor effect of the supernatant of lymphocytes is preserved.

Effect of the supernatant on the chemoluminescence of the platelets

The Inventors have demonstrated previously (C.R. ACAD. SC. PARIS, 1984,298:55 already mentioned) that, in the IgE-dependent toxicity relativeto schistosomules, it is possible to obtain the production ofmetabolites of oxygen by human platelets, measured by chemoluminescence.

The effect of the supernatent of T lymphocytes stimulated byConcanavaline A (1 μg/ml) on the chemoluminescence of the platelets hasbeen tested: as shown by accompanying FIG. 4, incubation of plateletswith lymphocyte T supernatant for one hour, at a final dilution of 1:4,considerably reduces (by 60%) the production of metabolites of oxygenwhich is normally observed in the case where the normal platelets havebeen stimulated by an IgE/anti-IgE reaction or in the case where theplatelets of allergic patients have been stimulated by the correspondingallergen, which confirms the direct role of the lymphokine according tothe invention, relative to the platelets themselves.

In FIG. 4, the initials NS and AS have the following meanings:

NS=incubated in normal serum,

AS=incubated in the serum of allegic patients,

AS+ConA Sup=incubated in the serum of allergic patients and thesupernatant of lymphocytes.

The maximum activity of the luminol/luciferin chemoluminescence of 5.10⁵platelets in PBS has been recorded in the five minutes which followedthe addition of the medium (1st column of each group) or of theretarding agent: anti-IgE (10 μg of antibody/ml) (2nd column of eachgroup) or allergen (10 μg/ml) (3rd column of each group).

Characterization of the cells involved in the production of suppressorlymphokine

To define the type cell responsible for the production of the inhibitorfactor according to the present invention, the Inventors have examinedthe effect of a depletion of the selective T subpopulation: they treatedT cells with monoclonal OKT4 antibodies (for the auxiliary/inductorsubclasses) or by OKT8 monoclonal antibody (for thesuppressor/destructor subclasses) in the presence of complement. Thecells were then stimulated by Concanavaline A and it was examinedwhether the suppressor lymphokine was produced.

As shown by FIG. 5, the depletion of OKT8⁺ lymphocytes suppressed theproduction of inhibitor factor, whilst the depletion of OKT4⁺ cells didnot modify its formation. FIG. 5 illustrates the level of respectivesuppressor activity of the LSPA obtained from T cells, from OKT8⁺ OKT4⁻cells and from OKT8⁻ OKT4⁺ cells, stimulated by Concanavaline A. Theresults are expressed in % suppression (mean) of the IgE-dependentcytotoxicity, with respect to schistosomules.

This observation shows indeed that a sub-population of suppressive Tcells produces the factor responsible for the inhibition observed of theeffector functions of the platelets.

Effect of the treatment of T cells with indomethacine on the productionof inhibitor lymphokine (LSPA) according to the invention

The prostaglandins play, as is known, a role in the induction ofnonspecific suppressive T cells, during the mitogenic and antigenicstimulation of lymphocytes. The Inventors have therefore treated thelymphocytes with indomethacine (10⁻⁵ M final concentration) which is aninhibitor of the synthesis of protaglandins and they have examined theproduction of inhibitor lymphokine induced by the mitogenic agent. Theproduction of this factor remained unchanged under these conditions(48.0±3.0% of suppressive activity with indomethacine, for 45.0±2.5%without indomethacine).

Physicochemical characteristics of platelet inhibitor lymphokine (LSPA)according to the invention

The suppressor effect remained unchanged after dialysis of thesupernatant of lymphocytes for 24 hours against a buffer solution ofphosphate (PBS). The inhibitor factor was stable with respect to heat(56° C. for one hour or 100° C. for 5 minutes) and acids, since dialysisfor 24 hours against a buffer at pH2 did not alter its suppressoreffect, as is seen from Table 3 below which shows the physicochemicalproperties of LSPA: the stability of LSPA with respect to heat wasevaluated by heating the supernatants on the water bath at 56° C. for120 minutes, or at 100° C. for 5 to 10 minutes. The dialysed LSPA wasprepared by dialysis of the supernatant at 4° C. for 24 hours againstPBS, with multiple changes of medium. The supernatants treated with acidwere prepared by dialysis at 4° C. for 24 hours against glycine bufferat pH2, followed by dialysis for 24 additional hours against PBS toremove the excess acid.

                  TABLE 3                                                         ______________________________________                                        Physicochemical properties of PSPA                                                                Inhibition index                                          Treatment           %                                                         ______________________________________                                        A.    Heating           64.0 ± 1.4                                               120 minutes at 56° C.                                                                    65.7 ± 5.6                                                5 minutes at 100° C.                                                                    74.8 ± 8.8                                         B.    Dialysis for 24 hours                                                                           64.3 ± 3.6                                         C.    Acid treatment for 24 hours                                                                     68.5 ± 2.5                                         ______________________________________                                    

As is shown by accompanying FIG. 6 which illustrates the percentageinhibition of the cytotoxicity of supernatants treated by variousenzymes, respectively trypsin, protease K and neuraminidase, lymphokineisolated according to the present invention is sensitive to trypsin andto protease K, whilst neuraminidase has no influence on its activity.

A chromatographic profile of the inhibitor lymphokine of bloodplatelets, according to the present invention, on a molecular sieve ofSephadex-G75, is shown in FIG. 7. The LSPA was fractionnated on aSephadex-G75 column prepared in phosphate buffer (pH 7.4). The elutedfractions were tested for their LSPA activity in anti-schistosome,IgE-dependent platelet cytotoxicity. The suppressive activity wasdetected in two peaks, but the optimal activity was found in the secondpeak, the smallest, migrating into the range of molecular weights lessthan or equal to 25,000 and preferably comprised between 18,000 and20,000; the first and largest peak could correspond to an agregated formof the molecule.

The electric charge of the LSPA platelet inhibitor lymphokine wasexamined: the activity was maximum at an isoelectric point (Pi)comprised between 4.1 and 4.6, as is shown by FIG. 8 which illustratesthe percentage inhibition of cytotoxicity as a function of pH. Focusingthe isoelectric point of the LSPA was performed in a column ofSephadex-G75 containing 5% of support ampholines in a pH range of 3.8 to10 for 16 hours, at a constant power of 8 watts. Each gel fraction waseluted, dialysed overnight against a PBS containing NaCl to remove theampholines, after which its suppressor activity was examined byevaluating its IgE-dependent platelet cytotoxicity with respect toSchistosomules.

It results from the foregoing that LSPA lymphokine inhibits the effectorfunctions of blood platelets, which suggests that it plays a role in theregulation of immunopathological disorders in which the IgE level isincreased, particularly in atopic allergies, and more particularly inasthma. Through this fact, LSPA constitutes an immunopharmacologicalsubstance of great interest for the treatment of allergic disorders inwhich the platelets seem to be involved.

The description which follows refers to an example of the preparation ofLSPA lymphokine according to the present invention, which is given byway of illustration of one of the objects of the present invention, andhas no limiting character.

Example of the preparation of purified LSPA lymphokine 1. Preparation ofT lymphocytes supernatants

The T lymphocyte supernatants can be obtained either by mitogenicstimulation, or by antigenic stimulation, as described below.

A. By mitogenic stimulation

from human cells

3.10⁶ peripheral or amygdal cells taken up from man, were incubated inRPMI-FCS [that is to say RPMI-1640 medium containing 5% of foetal calfserum inactivated by heat (FSC)] with Concanavaline A (0.01 at 5 μg/ml)at 37° C. in culture plates comprising cups with a flat bottom, for 24hours, in a moist atmosphere containing 5% of CO₂. Cells were thenwashed to remove the concanavaline A and cultivated for 24 hours. Thesupernatants of each cup were recovered, centrifuged at 400 xg andfiltered through membranes of porosity 0.22 μm, after which, if theywere not used immediately, were stored at -20° C.

from murine cells

1.10 lymphocytes of lymphatic ganglions were used and the supernatantsprepared as described above.

B. By antigenic stimulation

stimulation of human T cells coming from a patient afflicted withEchinicoccosis was carried out in RMPI-FCS medium by the addition ofcrude extracts of Echinococcus granulosus (final concentration: 5,40 or100) in the presence of irradiated (2000 rads, 2 minutes) autologousperipheral blood mononuclear cells (MCPB). After having dwelt for 24hours in a moist atmosphere, the cells were washed to remove theaforesaid extracts and they were cultured for 4 days. The culturesupernatant was recovered, filtered through a membrane of 0.22 μm andstored at -20° C. if not used immediately,

the T cells obtained from lymphatic ganglions are rats infected with S.mansoni (14, 35 or 56 days of infection) were incubated in RPMI-FCSmedium supplemented with IL2 (Interleukine 2), irradiated thymus cellsof the rat (APC) and antigen of the S. mansoni adult worm (50 μg/ml offinal concentration) in culture plates with multiple cups with a flatbottom, in a moist atmosphere containing 5% CO₂. This stimulation wasperformed every 5 days and the 15th day, the supernatants wererecovered, centrifuged to eliminate therefrom all contaminated cellspossibly present, and filtered through membranes of 0.22 μm, beforebeing stored at -20° C., if necessary. At the same time, the growthresponse of the T cells was measured under these conditions, after apulse of 16 hours with I μCi of H-Thymidine (1Ci/mmole), theincorporated radioactivity being determined by filtration of the culturethrough Millipore membranes and counting of the filters in a liquidscintillation fluid, in a beta-spectrometer.

2. Purification of supernatants to extract therefrom the activelymphokine (LSPA)

2.1 The supernatant, if necessary defrosted after storage, was dialysedin an Amicon cell (UM5 membrane) to remove therefrom the salts and thesmall molecules.

2.2 After dialysis, the LSPA lymphokine is purified by gel filtration ona column of extra fine Biogel P30 (granulometry 400 mesh) in a 1% AcOHmedium, at a linear flow rate of 2 cm/hour. The effluents control iseffected by reading at 254 nm and by researching the biological orimmunological activity as described below. The fractions thus localisedwere combined and freeze dried.

2.3 The protein (LSPA lymphokine) is then subjected to reverse phasechromatography (HPLC) on a bonded silica gel (such as octadecylsilane,for example) whose particles are of controlled granulometry (5 um) andwhose pores are calibrated (300 Å). The elution was performed by meansof CH₃ CN--H₂ O ranging from 1-99 to 60-40, in 3 hours. To these twophases was added 1% of trifluoroacetic acid for ionic matching.

The detection was done by optical density recording at 215 nm anddetermination of the biological activity. The fractions so located werethen freeze dried and checked: they were constituted by LSPA lymphokine.

3. Detection of biological activity

The biological activity was detected by the aptitude of the platelets tokill schistosome larvae in the presence of specific IgEs, and byevaluation of the chemoluminescence induced by the oxidating metabolismin the presence of luminol and luciferin.

4. Determination of molecular weight of LSPA lymphokine according to theinvention

The supernatant stimulated by Concanavaline A, in the example described,concentrated (2 ml), was filtered through a Sephadex G-75 column (1.8×38cm) and diluted with PBS at the flow rate of 5 ml/hour. The lymphokinicactivity of each fraction was tested as described in 3. above.

To determine the molecular weight of the lymphokine, the chromatogramwas calibrated with a kit for calibration of low molecular weights(supplied by PHARMACIA, UPPSALA, Sweden) containing bovine serum-albumin(BSA, m.w. 67000), of ovalbumin (m.w. 43000), chymotrysinogen A (m.w.25000) and ribonuclease A (m.w. 13700). The molecular weight of thepolypeptide which constitutes the lymphokine, determined as indicatedabove, was comprised between 15000 and 20000.

Thus as emerges from the foregoing there was isolated, according to thepresent invention, a lymphokine which is distinguished from lymphokinesidentified until now, both by its biological properties and by itsphysico-chemical properties, and which inhibits the effector functionsof platelets, thus providing an immunopharmacological substance adaptedto play an important role in the regulation of immunopathologicaldisorders in which the IgE level is increased, and more particularly inatopic allergies and more especially still in asthma.

We claim:
 1. An essentially pure lymphokine, wherein said lymphokine:a)is obtained from the supernatant of T OKT8⁺ lymphocyte cultures aftermitogenic or antigenic stimulation; b) inhibits IgE-dependent plateletcytotoxicity with respect to young larva of Schistosomia mansoni; c)inhibits platelet activation in non-IgE dependent intolerances; d) has amolecular weight in the range of about 15-20 kDa; and e) has a pI ofabout 3.7-5.0.
 2. The lymphokine of claim 1, wherein said lymphokine isobtained from the supernatant of T OKT8⁺ lymphocyte culture which havebeen stimulated by a mitogen or an antigen.
 3. The lymphokine of claim2, wherein said stimulating antigen is selected from the groupconsisting of antigens of Echinococcus granulosus and antigens ofSchistosomia mansoni.
 4. The lymphokine of claim 1, wherein saidlymphokine has a pI of about 3.70-4.68.
 5. The lymphokine of claim 1,wherein said lymphokine has a pI of 4.1-4.6.
 6. The lymphokine of claim1, wherein said lymphokine is stable to heating at 56° C. for 1 hour or100° C. for 5 minutes.
 7. The lymphokine of claim 1, wherein saidlymphokine is stable to dialysis at 4° C. for 24 hours at pH=2.
 8. Thelymphokine of claim 1, wherein the inhibitory activity of saidlymphokine is stable to treatment with neuraminidase, and saidinhibitory activity is decreased by treatment with trypsin and proteaseK.
 9. The lymphokine of claim 1, where in said lymphokine adsorbs to thesurface of platelets.
 10. A process for the preparation of thelymphokine of claim 1, comprising the steps of:a) stimulating andcultivating T-cells comprising OKT8⁺ lymphocytes with a mitogen or withan antigen selected from the group consisting of antigens ofEchinococcus granulosus and antigens of Schistosomia mansoni; b)recovering the culture supernatants, clarifying the supernatants bycentrifugation and filtration through a membrane having a pore dimensionof about 0.22 μm; c) fractionating the filtrate from said membrane bygel filtration on a molecular sieve; and d) recovering the fractionsfrom said molecular sieve which inhibit platelet activation, e)submitting recovered fractions from step d) to reverse phasechromatography on a bonded silica gel of 5 μm particle size and a porediameter of about 300 angstroms and eluted during about 3 hours with aCH₃ CN--H₂ O eluate gradient, ranging from a ratio of 1/99 to 60/40,said eluate containing 1% trifluoroacetic acid, and recovering thefractions from said chromatography which inhibit platelet activation.